Transcription is one of the central points of regulation of cellular functions. Understanding molecular mechanisms of transcription and its regulation remains one of the most important goals in biology. DNA-dependent RNA polymerases (RNAP), the enzymes capable of faithfully copying the information encoded in gene sequence into the mRNA product, are of central importance in the process of transcription. Our research is focused on bacterial and archaeal RNAP's. These enzymes are among the simplest multisubunit polymerases, offering the best chance for obtaining detailed molecular understanding of RNAP activity. Our long term goal is to understand the molecular mechanism by which RNA polymerase initiates transcription. Bacterial RNAP is an attractive target for new antibiotics since it is an essential enzyme. Detailed understanding of RNAP properties unique to the bacterial enzyme will aid the development of new antibiotics targeting RNAP. It is clear now that transcription initiation involves a complex interplay between numerous RNAP-promoter contacts. While we are beginning to understand how RNAP utilizes these contacts in transcription initiation reaction, many gaps in knowledge will need to be filled before a comprehensive understanding of transcription initiation is achieved. In this project we will focus on filling in three of such gaps. In aim 1 we will determine the role of conserved nontemplate -7 thymine in promoter melting by bacterial RNA polymerase. In aim 2 we will determine the role of -35 conserved promoter element and the spacer connecting -10 and -35 promoter elements in promoter melting by bacterial RNA polymerase. In aim 3, we will investigate the interplay between RNAP interactions with the core and secondary promoter elements in promoter escape by bacterial RNA polymerase. Furthermore, we will also begin to investigate the mechanism of transcription initiation by archaeal RNA polymerase (aim 4). Archaeal RNA polymerase exhibits eukaryote-like structure but bacteria-like promoter melting (i.e. no auxiliary ATP-dependent activities are necessary for melting). Thus, the studies with archaeal polymerase will provide insights into the evolution of transcription initiation mechanism and will facilitate understanding of general mechanistic features this process. In our studies we will utilize a combination of fluorescence techniques together with mutagenesis, kinetic, thermodynamic and structural analysis of promoter melting reactions. Upon completion of these studies mechanistic insights to a fundamental biological process will be gained. PUBLIC HEALTH RELEVANCE: This project will provide new insights into the mechanism of transcription initiation by bacterial RNA polymerase. Such information will enable development of new antibiotics targeting bacterial RNA polymerase. Development of new antibiotics targeting new targets is essential to combating emerging antibiotic-resistant strains.